Elucidating the genetic basis of adaptation is one of the primary goals of evolutionary biology. Two major unresolved questions are the number of genes involved in adaptations, and the relative contributions of changes in gene expression and changes in protein structure to phenotypic change. Ethanol is a natural constituent of breeding sites of Drosophila melanogaster, and resistance to ethanol increases strongly with latitude on different continents, indicating that the trait is under natural selection. The genetic basis of the temperate-tropical difference is largely unknown, however. The availability of powerful genetic tools for D. melanogaster and the wealth of background information on the Drosophila-ethanol system make it an excellent choice for investigating the genetic basis of a natural adaptation. The principal hypothesis to be tested in the proposed work is that adaptation to high ethanol levels in temperate populations has been brought about in part by cis-regulatory evolution. Differences in gene expression between temperate and tropical populations will be measured using microarrays and real-time PCR, and compared to differences resulting from artificial selection for increased resistance. To the extent that expression differences contribute to the natural geographic variation in ethanol resistance, one or more genes showing consistently higher (lower) expression in temperate than tropical populations will consistently increase (decrease) in expression as a result of selection for resistance. For genes meeting this criterion, we will use allele-specific expression assays to measure the contribution of cis-regulatory variation to the expression differences, and will estimate the effects of the genes on resistance in a quantitative trait locus (QTL) mapping experiment. If, for a given gene, the temperate/tropical and selected/control expression differences both have cis-regulatory components, and the gene is close to a QTL for resistance, the results will give strong evidence that the gene is a target of selection by ethanol. For such genes, expression will be manipulated using knockout mutations and the GAL4-UAS system to confirm their role in resistance. Alcohol abuse is a major public health problem. The results of this study are likely to give insights into alcohol's neurological and physiological effects, possibly identifying new targets for ethanol in the nervous system. These insights could potentially be used to develop drug or other treatments for alcoholism.